The waterbodies of the halo-volcanic Dallol complex: earth analogs to guide us, where to look for life in the universe.

Microbes are the Earth life forms that have the highest degree of adaptability to survive, live, or even proliferate in very hostile environments. It is even stated that microbes can cope with any extreme physico-chemical condition and are, therefore, omnipresent all over the Earth: on all the continents, inside its crust and in all its waterbodies. However, our study suggests that there exists areas and even water rich environments on Earth where no life is possible. To support the fact that water rich environments can be lifeless, we performed an extensive survey of 10 different hyper extreme waterbodies of the halo-volcanic Dallol complex (Danakil depression, Ethiopia, Horn of Africa). In our study, we combined physico-chemical analyses, mineralogical investigations, XRD and SEM-EDX analyses, ATP measurements, 16S rDNA microbial community determinations, and microbial culturing techniques. According to our findings, we suggest that the individual physico-chemical parameters, water activity, and kosmo-chaotropicity, are the two most important factors that determine whether an environment is lifeless or capable of hosting specific extreme lifeforms. Besides, waterbodies that contained saturated levels of sodium chloride but at the same time possessed extreme low pH values, appeared to be poly-extreme environments in which no life could be detected. However, we clearly discovered a low diversity microbial community in waterbodies that were fully saturated with sodium chloride and only mildly acidic. Our results can be beneficial to more precisely classify whole or certain areas of planetary bodies, including water rich environments, as either potentially habitable or factual uninhabitable environments.

Phenotypic and Genetic Characterization of Temperature-Induced Mutagenesis and Mortality in Cupriavidus metallidurans.

Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C, a phenomenon coined "temperature-induced mortality and mutagenesis" (TIMM). To scrutinize this aberrant phenotype further, the contributions of specific inducers and protective agents were determined. Different growth media, including lysogeny broth (LB) and Schatz, and components, including casamino acids, in particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well conserved within the C. metallidurans species, multiple and strain-specific TIMM inducers exist. Twenty-nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high-concentration nitrogen-containing media at 37°C.

DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance.

Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver as antimicrobial agent. Furthermore, increased resistance to metals may be accompanied by increased antibiotic resistance. Inactivation of the histidine kinase and concomitant upregulation of the cognate response regulator (RR) of the AgrRS two-component system was previously shown to play an important role in the increased silver resistance of laboratory adapted mutants of Cupriavidus metallidurans. However, binding of AgrR, a member of the OmpR/PhoP family of RRs with a conserved phosphoreceiver aspartate residue, to potential target promoters has never been demonstrated. Here we identify differentially expressed genes in the silver-resistant mutant NA4S in non-selective conditions by RNA-seq and demonstrate sequence-specific binding of AgrR to six selected promoter regions of upregulated genes and divergent operons. We delimit binding sites by DNase I and in gel copper-phenanthroline footprinting of AgrR-DNA complexes, and establish a high resolution base-specific contact map of AgrR-DNA interactions using premodification binding interference techniques. We identified a 16-bp core AgrR binding site (AgrR box) arranged as an imperfect inverted repeat of 6 bp (ATTACA) separated by 4 bp variable in sequence (6-4-6). AgrR interacts with two major groove segments and the intervening minor groove, all aligned on one face of the helix. Furthermore, an additional in phase imperfect direct repeat of the half-site may be observed slightly up and/or downstream of the inverted repeat at some operators. Mutant studies indicated that both inverted and direct repeats contribute to AgrR binding in vitro and AgrR-mediated activation in vivo. From the position of the AgrR box it appears that AgrR may act as a Type II activator for most investigated promoters, including positive autoregulation. Furthermore, we show in vitro binding and in vivo activation with dephosphomimetic AgrR mutant D51A, indicating that unphosphorylated AgrR is the active form of the RR in mutant NA4S.

Spontaneous mutation in the AgrRS two-component regulatory system of Cupriavidus metallidurans results in enhanced silver resistance.

The uncontrolled and widespread use of (nano)silver compounds has led to the increased release of these compounds into the environment, raising concerns about their negative impact on ecosystems. Concomitantly, silver resistance determinants are widely spread among environmental and clinically relevant bacteria although the underlying mechanisms are not yet fully understood. We show that Cupriavidus metallidurans is able to adapt to toxic silver concentrations. However, none of the known silver resistance determinants present in C. metallidurans are involved in the adaptative response. Instead, increased silver resistance is achieved by the concerted action of a two-component system AgrR-AgrS, previously not associated with metal resistance, and two periplasmic proteins PrsQ1 and PrsQ2. Both proteins belong to an unique group of small, uncharacterized, secreted proteins restricted to the genera Cupriavidus and Ralstonia. This system gives C. metallidurans the ability to withstand much higher silver concentrations. The latter could be facilitated by the accumulation of silver ions and the formation of silver nanoparticles.

Genomic and Transcriptomic Changes that Mediate Increased Platinum Resistance in Cupriavidus metallidurans.

The extensive anthropogenic use of platinum, a rare element found in low natural abundance in the Earth's continental crust and one of the critical raw materials in the EU innovation partnership framework, has resulted in increased concentrations in surface environments. To minimize its spread and increase its recovery from the environment, biological recovery via different microbial systems is explored. In contrast, studies focusing on the effects of prolonged exposure to Pt are limited. In this study, we used the metal-resistant Cupriavidus metallidurans NA4 strain to explore the adaptation of environmental bacteria to platinum exposure. We used a combined Nanopore⁻Illumina sequencing approach to fully resolve all six replicons of the C. metallidurans NA4 genome, and compared them with the C. metallidurans CH34 genome, revealing an important role in metal resistance for its chromid rather than its megaplasmids. In addition, we identified the genomic and transcriptomic changes in a laboratory-evolved strain, displaying resistance to 160 µM Pt4+. The latter carried 20 mutations, including a large 69.9 kb deletion in its plasmid pNA4_D (89.6 kb in size), and 226 differentially-expressed genes compared to its parental strain. Many membrane-related processes were affected, including up-regulation of cytochrome c and a lytic transglycosylase, down-regulation of flagellar and pili-related genes, and loss of the pNA4_D conjugative machinery, pointing towards a significant role in the adaptation to platinum.

Unintentional Genomic Changes Endow Cupriavidus metallidurans with an Augmented Heavy-Metal Resistance.

For the past three decades, Cupriavidus metallidurans has been one of the major model organisms for bacterial tolerance to heavy metals. Its type strain CH34 contains at least 24 gene clusters distributed over four replicons, allowing for intricate and multilayered metal responses. To gain organic mercury resistance in CH34, broad-spectrum mer genes were introduced in a previous work via conjugation of the IncP-1ß plasmid pTP6. However, we recently noted that this CH34-derived strain, MSR33, unexpectedly showed an increased resistance to other metals (i.e., Co2+, Ni2+, and Cd2+). To thoroughly investigate this phenomenon, we resequenced the entire genome of MSR33 and compared its DNA sequence and basal gene expression profile to those of its parental strain CH34. Genome comparison identified 11 insertions or deletions (INDELs) and nine single nucleotide polymorphisms (SNPs), whereas transcriptomic analysis displayed 107 differentially expressed genes. Sequence data implicated the transposition of IS1088 in higher Co2+ and Ni2+ resistances and altered gene expression, although the precise mechanisms of the augmented Cd2+ resistance in MSR33 remains elusive. Our work indicates that conjugation procedures involving large complex genomes and extensive mobilomes may pose a considerable risk toward the introduction of unwanted, undocumented genetic changes. Special efforts are needed for the applied use and further development of small nonconjugative broad-host plasmid vectors, ideally involving CRISPR-related and advanced biosynthetic technologies.

Cupriavidus metallidurans Strains with Different Mobilomes and from Distinct Environments Have Comparable Phenomes.

Cupriavidus metallidurans has been mostly studied because of its resistance to numerous heavy metals and is increasingly being recovered from other environments not typified by metal contamination. They host a large and diverse mobile gene pool, next to their native megaplasmids. Here, we used comparative genomics and global metabolic comparison to assess the impact of the mobilome on growth capabilities, nutrient utilization, and sensitivity to chemicals of type strain CH34 and three isolates (NA1, NA4 and H1130). The latter were isolated from water sources aboard the International Space Station (NA1 and NA4) and from an invasive human infection (H1130). The mobilome was expanded as prophages were predicted in NA4 and H1130, and a genomic island putatively involved in abietane diterpenoids metabolism was identified in H1130. An active CRISPR-Cas system was identified in strain NA4, providing immunity to a plasmid that integrated in CH34 and NA1. No correlation between the mobilome and isolation environment was found. In addition, our comparison indicated that the metal resistance determinants and properties are conserved among these strains and thus maintained in these environments. Furthermore, all strains were highly resistant to a wide variety of chemicals, much broader than metals. Only minor differences were observed in the phenomes (measured by phenotype microarrays), despite the large difference in mobilomes and the variable (shared by two or three strains) and strain-specific genomes.

Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry.

Remains of a medieval foundry were excavated by archaeologists in 2013 in Verdun (France). Ancient workshops specialized in brass and copper alloys were found with an activity between 13th to 16th c. Levels of Cu, Zn and Pb reached 20000, 7000 and 6000 mg kg-1 (dw), respectively, in several soil horizons. The objective of the present work was to examine the microbial community in this contaminated site. A total of 8-22 106 reads were obtained by shotgun metagenomics in four soil horizons. Bioinformatic analyses suggest the presence of complex bacterial communities dominated by Proteobacteria. The structure of the community was not affected by metals, contrary to the set of metal-resistance genes. Using selective media, a novel strain of Cupriavidus necator (eutrophus), strain B9, was isolated. Its genome was sequenced and a novel metal resistance gene cluster with Hg resistance genes (merRTPCA) followed by 24 copper-resistance genes (actP, cusCBAF, silP, copK1, copH4QLOFGJH3IDCBARS, copH2H1, copK2) was found. This cluster is partly homologous to the cop genes of Cupriavidus gilardii CR3 and C. metallidurans CH34. Proteomics indicated that the four copH genes were differentially expressed: CopH1 and CopH2 were mostly induced by Cd while CopH4 was highly expressed by Cu.

Draft Genome Sequences of Ralstonia pickettii Strains SSH4 and CW2, Isolated from Space Equipment.

Ralstonia pickettii SSH4 and CW2 were isolated from space equipment. Here, we report their draft genome sequences with the aim of gaining insight into their potential to adapt to these environments.

Genome Sequences of Cupriavidus metallidurans Strains NA1, NA4, and NE12, Isolated from Space Equipment.

Cupriavidus metallidurans NA1, NA4, and NE12 were isolated from space and spacecraft-associated environments. Here, we report their draft genome sequences with the aim of gaining insight into their potential to adapt to these environments.

Genome Sequence of Cupriavidus metallidurans Strain H1130, Isolated from an Invasive Human Infection.

Cupriavidus metallidurans H1130 was repeatedly isolated from different blood culture sets taken from a patient suffering from significant nosocomial septicemia. Here, we announce the H1130 genome sequence for use in comparative analyses and for exploring the adaptation and pathogenic potential of this bacterium.

Characterization of culturable Paenibacillus spp. from the snow surface on the high Antarctic Plateau (DOME C) and their dissemination in the Concordia research station.

Culturable psychrotolerant bacteria were isolated from the top snow on the high Antarctic Plateau surrounding the research station Concordia. A total of 80 isolates were recovered, by enrichment cultures, from two different isolation sites (a distant pristine site [75° S 123° E] and a site near the secondary runway of Concordia). All isolates were classified to the genus Paenibacillus by 16S rRNA gene phylogenetic analysis and belonged to two different species (based on threshold of 97 % similarity in 16S rRNA gene sequence). ERIC-PCR fingerprinting indicated that the isolates from the two different sites were not all clonal. All isolates grew well from 4 to 37 °C and were resistant to ampicillin and streptomycin. In addition, the isolates from the secondary runway were resistant to chromate and sensitive to chloramphenicol, contrary to those from the pristine site. The isolates were compared to 29 Paenibacillus isolates, which were previously recovered from inside the Concordia research station. One of these inside isolates showed ERIC- and REP-PCR fingerprinting profiles identical to those of the runway isolates and was the only inside isolate that was resistant to chromate and sensitive to chloramphenicol. The latter suggested that dissemination of culturable Paenibacillus strains between the harsh Antarctic environment and the inside of the Concordia research station occurred. In addition, inducible prophages, which are potentially involved in horizontal dissemination of genes, were detected in Paenibacillus isolates recovered from outside and inside the station. The highest lysogeny was observed in strains harvested from the hostile environment outside the station.

Variation in genomic islands contribute to genome plasticity in Cupriavidus metallidurans.

BACKGROUND: Different Cupriavidus metallidurans strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with C. metallidurans type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30. RESULTS: Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different C. metallidurans strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn4371-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of C. metallidurans CH34. CONCLUSIONS: Metal resistance determinants are shared by all C. metallidurans strains and their occurrence is apparently irrespective of the strain's isolation type and place. Cupriavidus metallidurans strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others.

Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation.

Cupriavidus metallidurans CH34 is a ß-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (ß-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.

Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants.

Phytoremediation of highly water soluble and volatile organic xenobiotics is often inefficient because plants do not completely degrade these compounds through their rhizospheres. This results in phytotoxicity and/or volatilization of chemicals through the leaves, which can cause additional environmental problems. We demonstrate that endophytic bacteria equipped with the appropriate degradation pathway improve the in planta degradation of toluene. We introduced the pTOM toluene-degradation plasmid of Burkholderia cepacia G4 into B. cepacia L.S.2.4, a natural endophyte of yellow lupine. After surface-sterilized lupine seeds were successfully inoculated with the recombinant strain, the engineered endophytic bacteria strongly degraded toluene, resulting in a marked decrease in its phytotoxicity, and a 50-70% reduction of its evapotranspiration through the leaves. This strategy promises to improve the efficiency of phytoremediating volatile organic contaminants.